Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin d metabolite

ABSTRACT

The invention concerns a process for the production of a hybridoma, and of a monoclonal antibody or fragments thereof able to recognize 25-hydroxyvitamin D 2  and 25-hydroxyvitamin D 3 .

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/255,164, filed Oct. 27, 2009, of PCT PatentApplication No. PCT/EP2009/064148, filed Oct. 27, 2009, and BelgianProvisional Patent Application No. BE 2010/0234, filed Apr. 12, 2010,the entirety of each which are hereby incorporated by reference intothis application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns a process for the production of a hybridoma and amonoclonal antibody obtained therefrom, able to recognize one epitopepresent on more than one antigen. In particular, the invention relatesto a process for the production of a hybridoma and a monoclonalantibody, or fragments thereof, able to recognize more than one vitaminD metabolites, namely 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃.

2. Description of Related Art

Vitamin D is the generic term used to designate vitamin D₂ orergocalciferol and vitamin D₃ or cholecalciferol. Humans naturallyproduce vitamin D₃ when the skin is exposed to ultraviolet sun rays.Vitamin D₃ is transferred to the liver, where it is metabolised into25-hydroxyvitamin D₃, which is the main form of vitamin D circulating inthe body. Since the nineteenth century, vitamin D₂ has been availableorally through food in order to compensate for a lack of vitamin D₃ forexample among people who are hardly exposed to sunlight. The oralconsumption of vitamin D₂ has become increasingly important over recentcenturies. In fact, it is currently known that vitamin D has a primaryrole in the body for calcium binding, and mineralization of bones. Italso plays a significant role in various metabolic pathways. The25-hydroxyvitamin D, and particularly 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ are the forms of vitamin D which are most easilyaccumulated in the body. These two precursors can be converted by thekidneys to form 1α,25-dihydroxyvitamin D, which is the biologicallyactive form. 1,25-dihydroxyvitamin D relates to the active forms ofvitamin D (also known as D hormones) which have a hydroxyl grouping inpositions 1 and 25 of the formula (A) and (B). More than fifty or sodifferent metabolites of vitamin D have been discovered to date. Amongthese are 24,25-dihydroxyvitamin D and 25,26-dihydroxyvitamin D.

It is important to ensure the content of vitamin D in the body can bemeasured. However, measuring the content of vitamin D is actually oflittle value, since vitamin D concentrations fluctuate significantlybased on the oral consumption of vitamin D₂. The same applies equally asregards the physiologically active forms of vitamin D(1α,25-dihydroxyvitamin D), which are also present in the body inrelatively low quantities and fluctuate significantly in comparison to25-hydroxyvitamin D. For these reasons, the quantification of25-hydroxyvitamin D (25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃) is avaluable tool to facilitate the overall analysis of vitamin D in anindividual. Various methods exist, some of which involve immunology, todetermine the presence of 25-hydroxyvitamin D.

Usually, immunological methods involve the use of polyclonal antibodies,or monoclonal antibodies. Polyclonal antibodies are known from a longtime. To obtain such polyclonal antibodies, animals (such as rabbits forinstance) need to be immunised with a highly purified preparation of anantigen. The immunisation has to be done repeatedly with such purifiedpreparation. However, most frequently, this leads to the production of amixture of antibodies, this mixture randomly binding more than oneantigen, thus leading to unspecific measurement. A first issue is thelife span of the host animal, Frequently, multiple animals needed to beimmunised because of biological variability, Some animals being thesource of the most specific antisera have to be selected and the deathof said animals lead to the end of the production of the desiredantibodies, until next immunisation of animals. Secondly, the antiserumreactivity is also a concern because the desired antibody is only afraction of the total antibodies in the serum, which is itself a tooheterogenic mixture. Further, the technique suffers from batch-to-batchvariability. However, the production of polyclonal antibodies is a quickand inexpensive technique which has been and is still widely used inimmunology.

In the field of recognition of vitamin D metabolites, a lot of documentsdescribing the use of polyclonal antibodies are known. For instance,Hollis et al. (Clinical chemistry, 1993, 39, 529-533), EP 0092004,Kobayashi et al. (Steroids, 1994, 59, 401-411), and Clemens et al.(Steroids, 1983, 42, 503-509) disclose the production of polyclonalantibodies and their use in vitamin D radioimmunoassay.

In WO 2007/039193, Roche Diagnostics GmbH and F. HOFFMANN-LA ROCHE AGdescribe various methods of the state of the art. The latter disclose aprocess to produce polyclonal antibodies against 25-hydroxyvitamin D,which includes steps of immunising an animal with a conjugate whichcontains 25-hydroxyvitamin D₃ or 25-hydroxyvitamin D₂ as a hapten,isolating the serum or plasma of this animal and purifying theantibodies contained in the serum or the plasma by immunosorption on acomplementary matrix, which includes 25-hydroxyvitamin D₂ when thehapten is 25-hydroxyvitamin D₃ or which includes 25-hydroxyvitamin D₃when the hapten is 25-hydroxyvitamin D₂. EAH-Sepharose has been used asthe preferred material for the immunosorption matrix. One major problemarising with this method is that the antibodies are polyclonal, namelythat new immunisations must be performed on an animal to produce them.Another problem is that if the method does indeed allow both25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ to be determined in asample, it is necessary to carry out two test batteries involvingpolyclonal antibodies to find the total amount of the two components of25-hydroxyvitamin D, i.e. one test to determine the amount of25-hydroxyvitamin D₂ in the sample and another one to determine theamount of 25-hydroxyvitamin D₃ in the same sample.

For the quantification of a molecule in biological samples, for instancein diagnostic kits, monoclonal antibodies can also be used. The Kohlerand Milstein's invention dated 1975 opened a totally new field inimmunology through a technique involving the fusion, in the presence ofpolyethylene glycol, of a myeloma cell rendered drug sensitive through amutation in gene HGPRT with immune spleen B cells from a host animalimmunised with the antigen of interest. Hybridoma cells survive and maybe cultured in an appropriate medium (HAT) and rendered immortal.Because each hybridoma descends from one B cell, it makes copies of onlyone monoclonal antibody. The hybridoma that produces the antibody ofinterest is grown in culture to produce large amounts of monoclonalantibody, which are then isolated for further use. It is worth notingthat monoclonal antibody is known to be highly specific antibodiesagainst one epitope.

In the field of recognition of metabolites of vitamin D, monoclonalantibodies are used too. For instance Perry et al. (Biochemical andbiophysical research communications, 1983, 112, 431-436), U.S. Pat. No.4,585,741 and Kobayashi et al. (Biol. Pharm. Bull., 1997, 20(9),948-953) disclose the use monoclonal antibodies against 1,25dihydroxycholecalciferol. It is worth noting that the normal productionscheme of a monoclonal antibody directed against one specific antigenincludes the use of one hapten, the immunisation of one animal(generally a mouse), that after fusion between spleen cell and myelomacell, one hybridoma is produced, and this latter may be cultured andimmortalized to produce one monoclonal antibody specifically directedagainst a single epitope on a single antigen.

If one skilled in the art would like to use monoclonal antibodies forthe detection of more than one antigen, he must produce in two parallelprocesses a monoclonal antibody for each molecule to be detected. Forexample WO 03/104820 discloses the quantification of vitamins A and D₃in fluid samples. A monoclonal antibody against vitamin A is producedusing standard procedures from vitamin A-KLH conjugate (i.e. the haptenis vitamin A). A monoclonal antibody against vitamin D₃ is produced fromvitamin D₃-KLH conjugate (i.e. the hapten is vitamin D₃). Hence, twotests have to be performed to obtain the amount of both vitamins in thefluid samples.

SUMMARY OF THE INVENTION

It has now been found that the process according to the invention allowsa monoclonal antibody produced by one and the same hybridoma to be ableto recognize one epitope present on more than one antigen, which iscontrary to all expectations in the art. In particular, the inventorshave found that both 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ binda monoclonal antibody issued from a single hybridoma.

For the sake of clarity, although it should be understood that, withinthe frame of the present invention, a given hybridoma produces a giventype of monoclonal antibody, i.e. in practice several molecules of thesame structure, in the present text, when speaking about the invention,one will use “monoclonal antibody” to designate the production ofmonoclonal antibody from a single hybridoma, and “monoclonal antibodies”to designate the production of monoclonal antibody from differenthybridomas.

Unless otherwise specified, the term “vitamin D” must be understoodwithin the context of the present document as including the forms ofvitamin D₂ and vitamin D₃ with the following formula (A) and (B):

In formula (A) and (B), the positions of vitamin D are reflected in thenomenclature of the steroids. 25-hydroxyvitamin D indicates the vitaminD metabolites which are hydroxylated at position 25 of the formula (A)and (B), namely 25-hydroxyvitamin D₂ as well as 25-hydroxyvitamin D₃. Asabove-mentioned, 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ areparticularly relevant forms of vitamin D when used for diagnosticpurposes.

The vitamin D metabolites are not immunogen as such. The chemicalactivation of the components resulting from the metabolism of vitamin Das well as their binding to carrier proteins or linked groups issignificant. Therefore, to ensure successful immunisation, it is vitalto prepare a conjugate which may contain a metabolite of vitamin D or aderivative thereof as a hapten. The term “a hapten” must be understoodby a person skilled in the art as a substance which is not immunogenicin itself, but which, by coupling with a carrier protein, is revealed ina form against which antibodies can be generated. Carrier proteins forthe production of conjugates of haptens, i.e. immunogens, are known tothose of ordinary skill in the art. The bovine serum albumin (BSA), theβ-galactosidase or the keyhole limpet hemocyanine (KLH) are currentlyused as carrier proteins. The term “carrier protein” refers to a proteinwhich transports a specific substance or a group of substances thoughthe cellular membrane, in extra-cellular fluids or in an intracellularcompartment.

Only position 3 of the structures such as represented in formula (A) and(B) is, in principle, suitable for the activation and coupling of thecarrier proteins. In fact, the vitamin D metabolites are believed tobind via position 3 (see WO 2007/039194; Kobayashi and al. “Productionand specificity of antisera raised against 25-hydroxyvitaminD₃-[C-3]-bovine serum albumin conjugates”, Steroids 1992, 57(10), pp.488-493).

According to a first aspect, the present invention relates to a processfor the production of a hybridoma, and of a monoclonal antibody, orfragments thereof, able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃, comprising the following steps:

a) Immunisation of an animal with a hapten, rendered immunogenic, ofgeneral formula (I), or a salt thereof, or a derivative thereof in whichthe carboxylic acid function is protected to form an ester, amide oroxazoline,

wherein

n is an integer between 0 and 3;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁸ are independentlyhydrogen or a C₁₋₄ alkyl;

R¹² and R¹³ are a C₁₋₄ alkyl;

R is a hydrogen or a substituent selected from the group consisting of agroup of acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl,methoxytrityl, tetrahydropyranyl, triphenylmethyl groups, and a silylderivative;

b) sampling of B cells produced by the animal and fusion thereof withmyeloma cells to form the hybridoma;

c) production, from at least part of the obtained hybridoma, of amonoclonal antibody, or fragments thereof, able to recognize25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃.

The present process allows to produce a monoclonal antibody able to bindor recognize 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ (twodifferent molecules) from a hybridoma obtained by immunisation with asingle hapten renders immunogenic, i.e. with a single conjugate. This isa totally unexpected result which could not be suggested by prior art.Indeed, as previously mentioned, it is known in the art that amonoclonal antibody specifically binds one antigen which is similar tothe hapten used. Herein, the present invention surprisingly discloses amonoclonal antibody binding two different antigens, said monoclonalantibody being produced by a hybridoma provided by immunisation with asingle hapten of general formula (I).

The hapten of general formula (I) may be in the form of a carboxylatesalt. Organic or Inorganic salts may be convenient. Therefore, the saltsused can be, for example, sodium, potassium or ammonium salts.Alternatively, the hapten of general formula (I) may be a derivative inwhich the carboxylic acid function may be protected to form an ester,amide or oxazoline. The ester may be an alkyl-, aryl-, benzylic-, thio-,seleno-, silyl- or ortho-ester.

In the present invention, the term “C₁₋₄alkyl” refers to a hydrocarbonradical having the formula C_(m)H_(2m+1) in which m is an integerbetween 1 and 4. For example the term “C₁₋₄alkyl” refers to methyl,ethyl, n-propyl, i-propryl, n-butyl, i-butyl, s-butyl and t-butylradical. The term “acyl” refers to a radical of formula T¹C(O)— in whichT¹ is a substituent alkyl or aryl. The term “benzyl” refers to a radicalof formula T²CH₂— in which T² is an aryl group. The term “alkyl ether”refers to a radical of formula T³OT⁴- in which T³ is an alkyl, an arylor a benzyl and T⁴ a hydrocarbon chain of formula —(CH₂)_(p)— in which pis an integer between 1 and 10. The term “dimethoxytrityl” refers to theradical bis-(4-methoxyphenyl)phenylmethyl. The term “methoxytrityl”refers to the radical 4-methoxyphenyl)diphenylmethyl.

The term “aryl” according to the present invention refers to an aromatichydrocarbon, polyunsaturated group, with one or more fused rings (forexample napthyl) or covalently bound, generally containing between 6 and10 carbon atoms, in which at least one cycle is aromatic. The rings canbe substituted. Non-exhaustive examples include phenyl, napthyl,anthracyl and biphenyl groups.

The term “substituted” as used in the present invention indicates thatone or more hydrogens of the atom indicated in the expression using“substituted” is replaced with a selection from the Indicated group,subject to the valency of the atom(s) indicated not exceeding the normalvalency of the same, and that the substitution results in a chemicallystable compound, namely a compound sufficiently robust to survive in aclearly identifiable form and to an acceptable degree of purity from thereaction mixture. Substituents can be selected, but not limited to, fromthe group consisting of alkyl, aryl, cycloalkyl, halide, hydroxyl,nitro, amido, carboxy, amino and cyano groups. In the present invention,the term “nitro” refers to the group —NO₂. In the present invention, theterm “cyano” refers to the group —CN. In the present invention, the term“hydroxyl” refers to the group —OH. In the present invention, the term“amido” refers to the group —C(O)—NH—. In the present invention, theterm “carboxy” refers to the group —C(O)O—. In the present invention,the term “halide” refers to the chloride, fluoride, bromide and iodideradicals. In the present invention, the term “amino” refers to theradical of a trivalent nitrogen atom substituted or not. In the presentinvention, the term “cycloalkyl” refers to a cyclic alkyl groupincluding all the hydrocarbon groups containing one or two rings,including monocyclic or bicyclic groups. The cycloalkyls include atleast three carbon atoms in the cycle, preferably between 3 and 10carbon atoms and can be optionally substituted.

The term “alkyl” refers to a radical hydrocarbon of formulaC_(m)H_(2m+1) in which m is an integer greater than 1. Generally, thealkyl groups of the present invention include between 1 and 10 carbonatoms. For example, the term “C₁₋₁₀ alkyl” refers but is not limited toa radical methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl,t-butyl, 1-pentyl, 2-pentyl, 3-pentyl, i-pentyl, neo-pentyl, t-pentyl,1-hexyl, 2-hexyl, 3-hexyl, 1-methyl-1-ethyl-n-pentyl,1,1,2-tri-methyl-n-propyl, 1,2,2-trimethyl-npropyl,3,3-dimethyl-n-butyl, 1-heptyle, 2-heptyle,1-ethyl-1,2-dimethyl-n-propyl, 1-ethyl-2,2-dimethyl-n-propyl, 1-octyle,3-octyle, 4-methyl-3-n-heptyle, 6-methyl-2-n-heptyle,2-propyl-1-n-heptyle, 2,4,4-trimethyl-1-n-pentyl, 1-nonyle, 2-nonyle,2,6-dimethyl-4-n-heptyle, 3-ethyl-2,2-dimethyl-3-n-pentyl,3,5,5-trimethyl-1-n-hexyl, 1-decyle, 2-decyle, 4-decyle,3,7-dimethyl-1-n-octyle, 3,7-dimethyl-3-n-octyle. The alkyl group may besubstituted.

The term “tetrahydropyranyl” refers to a radical of 2-tetrahydropyrane.

The term “triphenylmethyl” refers to a radical methyl substituted bythree aryl groups, preferably phenyl.

The term “silyl derivative” refers to a radical of formula T⁵T⁶T⁷Si— inwhich T⁵, T⁶, T⁷ are independently an alkyl, aryl, alkoxy and aryloxy.In the present invention, the term “alkoxy” refers to a radical offormula —OT⁸ in which T⁸ is an alkyl substituted or not. In the presentinvention, the term “aryloxy” refers to a radical of formula —OT⁹ inwhich T⁹ is an aryl substituted or not.

Said hapten may be rendered immunogen by a covalent coupling with animmunogenic carrier protein, by encapsulation in the liposomes, byanchorage in the liposomes, by coupling of said hapten with a polymer,by induction with a biopolymer, or by coupling with a multiple antigenicpeptide. The term “immunogen” refers to a substance likely to trigger animmune reaction. Unlike the immunogen, the hapten is a derivative whichcan be identified by the immune system (for example antibodies) butwhich does not trigger any immune reaction.

When said hapten is covalently bound with an immunogenic carrierprotein, said carrier protein may be BSA (bovine serum albumin),ovalbumin, HSA (human albumin serum), THY (thyroglobulin), KLH (keyholelimpet hemocyanine), cBSA (bovine cationic albumin), β-galactosidase orCCH (Concholepas hemocyanine).

When said hapten is bonded to a polymer, said polymer may be asynthetic, natural or modified natural polymer. Therefore, the syntheticpolymer may be, but not limited to, for example poly-L-lysine oragarose. Alternatively, the natural polymer maybe, but not limited to,dextrane. Alternatively, the modified natural polymer may be, but notlimited to, carboxymethyl cellulose.

Alternatively, said hapten may be rendered immunogenic by induction witha biopolymer. The process is described in the publication Basalp and al.“Immunogenic Cu²⁺-induced Biopolymer systems comprising a steroidhormone, protein antigen, and synthetic polyelectrolytes (Hybridoma andHybridomics, 2002, 21(1), pp. 45-51).

Alternatively, said hapten may be bonded to a multiple antigenicpeptide. Said multiple antigenic peptides may be a polylysine core towhich 2 to 16 copies of a synthetic peptide are bonded.

Preferably, said hapten is rendered immunogenic by a covalent couplingwith a carrier protein. The KLH and BSA may be particularly effectivecarrier proteins for the process and use of the present invention. Thecoupling between the hapten and the carrier protein may be performed viathe carboxylic acid function of the hapten.

Preferably, the hapten may be a derivative of general formula (I) inwhich n is equal to 0. In particular, said hapten may be the derivativeof general formula (I), in which n is equal to 0 and in which R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl group. Therefore, the name of suchhapten according to the IUPAC standard is the(2S)-2-((7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)propanoicacid. This compound can also be named acid23,24,25,26,27-pentanor-9,10-secocholesta-5,7,10(19)-trien-3β-ol-22-oic,the CAS number of which is 99518-38-4.

Alternatively, the hapten may be a derivative of general formula (I) inwhich n is equal to 1. In particular, said hapten may be the derivativeof general formula (I) in which n is equal to 1 and in which R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl group. Therefore, the name of suchhapten according to the IUPAC standard is the acid(R)-3-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)-ethylidene)-7a-methyloctahydro-1H-inden-1-yl)butanoic.This compound can also be named acid24,25,26,27-tetranor-9,10-secocholesta-5,7,10(19)-trien-3β-ol-23-oic,the CAS number of which is 76794-34-8.

Alternatively, the hapten may be a derivative of general formula (I) inwhich n is equal to 2. In particular, said hapten may be the derivativeof general formula (I) in which n is equal to 2 and in which R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl group. Therefore, the name of suchhapten according to the IUPAC standard is the acid(R)-4-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-pentanoic.This compound can also be named acid25,26,27-trinor-9,10-secocholesta-5,7,10(19)-trien-31-ol-24-oic.

Alternatively, the hapten may be a derivative of general formula (I) inwhich n is equal to 3. In particular, said hapten may be the derivativeof general formula (I) in which n is equal to 3 and in which R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl group. Therefore, the name of suchhapten according to the IUPAC standard is the acid(R)-5-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-hexanoic.This compound can also be named acid26,27-bisnor-9,10-secocholesta-5,7,10(19)-trien-3β-ol-25-oic.

Preferably, the hybridoma produced following the fusion of the myelomawith the B cells of the animal can be chosen in the group consisting ofthe hybridomas deposited in the BCCM/LMBP (BCCM/LMBP® BelgianCoordinated Collections of Microorganisms—Department of BiomedicalMolecular Biology—Ghent, Belgium) on Sep. 21, 2009 under deposit numbersLMBP 7011CB, LMBP 7012CB and LMBP 7013CB, and hybridomas deposited inthe BCCM/LMBP on Mar. 9, 2010 under deposit numbers LMBP 7205CB and LMBP7204CB. The animal used for the experiments may be a rabbit, mouse,hamster, rat, and others.

Each of the monoclonal antibody produced by a single hybridoma of thepresent process is able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. The recognition of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ may be simultaneous. The term “simultaneous” meansthat a monoclonal antibody produced by the process of the presentinvention is able to bind or recognize both 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ when both antigens are present in the same sample.Therefore, the recognition percentage of the 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ may be between 70 and 110%. The recognitionpercentage is the ratio, multiplied by 100, between the total quantityof 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ recognized by theantibody according to the invention and the total effective quantity of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ present in the sample tobe tested. The percentage may exceed 100%. This is due to theuncertainty associated with the method of measuring overall quantitiesof 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. This is a phenomenonwell known to those of ordinary skill in the art for this type ofmeasurement.

According to a second aspect of the invention, several hybridomas eachable to produce a monoclonal antibody or fragments thereof able torecognize 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ are provided. Ahybridoma may be obtainable by the steps of a) and b) of the presentprocess. The hybridoma can be chosen in the group consisting of thehybridomas deposited in the BCCM/LMBP (Belgian Coordinated Collectionsof Microorganisms—Department of Biomedical Molecular Biology—Ghent,Belgium) on Sep. 21, 2009 under deposit numbers LMBP 7011CB, LMBP 7012CBand LMBP 7013CB, and hybridomas deposited in the BCCM/LMBP on Mar. 9,2010 under deposit numbers LMBP 7205CB and LMBP 7204CB. As stated above,each of these hybridomas is able to produce a monoclonal antibody, orfragments thereof, able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. The recognition of the 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ by said monoclonal antibody, produced by ahybridoma according to the invention, may be simultaneous when thesample tested contains both 25-hydroxyvitamin D₂ and 25-hydroxyvitaminD₃. It is particularly remarkable that the monoclonal antibody, or thefragments thereof, produced by the hybridoma according to the inventionmay reveal a recognition percentage of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ ranging from 70 to 110%. Said hybridoma may be usedin the manufacturing of a diagnostic device able to recognize andquantify 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in a sample orsamples to be tested or may be used for the production of monoclonalantibody or fragments thereof able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. Said hybridoma may be genetically engineered forenhancing the secretion of monoclonal antibody produced therefrom, i.e.plasmid or DNA sequence may be added to the DNA sequence of thehybridoma to form a genetically engineered hybridoma which can be usedfor producing monoclonal antibody able to recognize 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃.

According to a third aspect of the invention, monoclonal antibody orfragments thereof is provided. Said monoclonal antibody or fragmentsthereof is able to recognize 25-hydroxyvitamin D₂ and 25-hydroxyvitaminD₃. Therefore, monoclonal antibody or fragments thereof against the twoantigens 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ are provided.Preferably, said monoclonal antibody may be obtainable by the process ofthe present invention. Preferably, said monoclonal antibody or fragmentsthereof can be produced from a hybridoma selected from the groupconsisting of the hybridomas deposited in the BCCM/LMBP under depositnumbers LMBP 7011CB, LMBP 7012CB and LMBP 7013CB, and hybridomasdeposited in the BCCM/LMBP on Mar. 9, 2010 under deposit numbers LMBP7205CB and LMBP 7204CB.

Each of these hybridomas is able to produce a monoclonal antibody, orfragments thereof, able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃.

The recognition, by said monoclonal antibody, of 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ may be simultaneous. Moreover, the recognitionpercentage of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ may bebetween 70 and 110%.

According to a fourth aspect of the invention, the invention concernsthe use of a hapten consisting of a compound of general formula (I), ora salt thereof, or a derivative thereof in which the carboxylic acidfunction is protected to form an ester, amide or oxazoline,

wherein

n is an integer between 0 and 3;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independentlyhydrogen or a C₁₋₄ alkyl;

R¹² and R¹³ are a C₁₋₄ alkyl;

R is a hydrogen or a substituent selected from the group consisting ofan acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl,methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silylderivative; said hapten rendered immunogenic;

for the production of a hybridoma, and of a monoclonal antibody orfragments thereof able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. Alternatively, the hapten consisting of a compoundof general formula (I) may be used for the production of recombinantsmonoclonal antibody fragments able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. Said hapten may be used for the production ofhybridoma according to steps a) and b) of the process of the presentinvention defined above. Said hapten may be used for the production of amonoclonal antibody according to the process of the present inventiondefined above.

Said hapten may be rendered Immunogenic by a covalent coupling with animmunogenic carrier protein, by encapsulation in the liposomes, byanchorage in the liposomes, by coupling of said hapten with a polymer,by induction with a biopolymer, or by coupling with a multiple antigenicpeptide.

When said hapten is covalently coupled with an immunogenic carrierprotein, said carrier protein may be the BSA (bovine serum albumin),ovalbumin, HSA (human serum albumin), THY (thyroglobulin), the KLH(Keyhole limpet hemocyanin), cBSA (cationic bovine serum albumin),β-galactosidase or CCH (Concholepas hemocyanine).

When said hapten is coupled to a polymer, said polymer may be asynthetic, natural or modified natural polymer. Therefore, the syntheticpolymer may be, but not limited to, for example poly-L-lysine oragarose. Alternatively, the natural polymer maybe, but not limited to,dextrane. Alternatively, the modified natural polymer may be, but notlimited to, carboxymethyl cellulose.

Alternatively, said hapten may be rendered immunogenic by induction witha biopolymer. The process is described in the publication Basalp and al.“immunogenic Cu²⁺-induced Biopolymer systems comprising a steroidhormone, protein antigen, and synthetic polyelectrolytes” Hybridoma andHybridomics, 2002, 21(1), 45-51, attached hereto in the reference.

Alternatively, said hapten may be coupled to a multiple antigenicpeptide. Said multiple antigenic peptides may be a polylysine core where2 to 16 copies of a synthetic peptide are coupled.

Preferably, said hapten is rendered immunogenic by a covalent bond witha carrier protein. The KLH and BSA can be particularly effective carrierproteins for the process and the use of the present invention. Thecoupling between the hapten and carrier protein may be performed via thecarboxylic acid function of the hapten.

Preferably, the hapten used for the production of a hybridoma andmonoclonal antibody obtained therefrom and able to recognize25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ may be a compound offormula (I) in which n is equal to 0. In particular, said hapten may bethe acid(2S)-2-((7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)propanoic,namely the compound of formula (I) in which n is equal to 0; R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl groups. Alternatively, the haptenused for the production of a hybridoma and monoclonal antibody obtainedtherefrom and able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ may be the compound of formula (I) in which n isequal to 1. In particular, the hapten may be the acid(R)-3-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclo-hexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)butanoic,namely the compound of formula (I) in which n is equal to 1; R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl groups. Alternatively, the haptenused for the production of a hybridoma and monoclonal antibody obtainedtherefrom and able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ may be the compound of formula (I) in which n isequal to 2. In particular, the hapten may be the acid(R)-4-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)-ethylidene)-7a-methyloctahydro-1H-inden-1-yl)pentanoic,namely the compound of formula (I) in which n is equal to 2; R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl groups. Alternatively, the haptenused for the production of a hybridoma and monoclonal antibody obtainedtherefrom and able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ may be the compound of formula (I) in which n isequal to 3. In particular, the hapten may be the acid(R)-5-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)-ethylidene)-7a-methyloctahydro-1H-inden-1-yl)hexanoic,namely the compound of formula (I) in which n is equal to 3; R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl groups.

Therefore, the recognition of 25-hydroxyvitamin D₂ and 25-hydroxyvitaminD₃ by said monoclonal antibody, or the fragments thereof, may besimultaneous when both antigens are present in the same sample.Moreover, the recognition percentage of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃, by said monoclonal antibody or the fragmentsthereof, may be between 70 and 110%.

The hapten and/or monoclonal antibody, or the fragments thereof,according to the invention can be used in the manufacturing of adiagnostic device able to recognize and quantify 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ in a sample or samples to be tested. The haptenbeing a compound of general formula (I) such as defined above, may beused in the manufacturing of a diagnostic device able to recognize andquantify 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in a sample to betested. In this case, the recognition of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ by said monoclonal antibody may be simultaneouswhen both antigens are in the same sample. The recognition percentage of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ by said monoclonalantibody may be between 70 and 110%.

According to another aspect of the invention, a diagnostic device likelyto allow the recognition and/or quantification of 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ in a sample to be tested is provided. The term“diagnostic device” as used herein also refers to a kit for therecognition and/or quantification of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ in a sample. The sample may be a human or animalsample. Diagnostic device encompasses testing device for animalresearch. Said diagnostic device includes a monoclonal antibody orfragments thereof able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃. Preferably, the monoclonal antibody or thefragments thereof of said diagnostic device may be produced, accordingto the present invention, from a hybridoma selected from the groupconsisting of the hybridomas deposited in the BCCM/LMBP under depositnumbers LMBP 7011CB, LMBP 7012CB, LMBP 7013CB, LMBP 7204CB and LMBP7205CB. The recognition of 25-hydroxyvitamin D₂ and 25-hydroxyvitaminD₃, by said monoclonal antibody or fragments thereof, may besimultaneous. Moreover, the recognition percentage of 25-hydroxyvitaminD₂ and 25-hydroxyvitamin D₃, by said monoclonal antibody or fragmentsthereof, may be between 70 and 110%. Said sample to be tested may be abiological sample from human or animal origin.

The diagnostic device may also include a sample of the compound offormula (I), or a salt thereof, or a derivative thereof in which thecarboxylic acid function is protected to form an ester, amide oroxazoline,

in which

n is an integer between 0 and 3;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁶ are independentlyhydrogen or a C₁₋₄ alkyl;

R¹² and R¹³ are a C₁₋₄ alkyl;

R is a hydrogen or a substituent selected from the group consisting ofan acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl,methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silylderivative.

Preferably, the diagnostic device may include a sample of the compoundof formula (I) in which n is an integer equal to 0; R, R², R³, R⁴, R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen; R¹² andR¹³ are respectively methyl groups. Alternatively, the diagnostic devicemay include a sample of the compound of formula (I) in which n is aninteger equal to 1; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴,R¹⁵ are respectively hydrogen; R¹² and R¹³ are respectively methylgroups. Alternatively, the diagnostic device may include a sample of thecompound of formula (I) in which n is an integer equal to 2; R, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen;R¹² and R¹³ are respectively methyl groups. Alternatively, thediagnostic device may include a sample of the compound of formula (I) inwhich n is an integer equal to 3; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen; R¹² and R¹³ arerespectively methyl groups.

The diagnostic device may also include means of expression of arepresentative signal of the presence of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ in a sample to be tested.

Said means of expression of a signal may be the tracer of formula (II),

wherein

n is an integer between 0 and 3;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independentlyhydrogen or a C₁₋₄ alkyl;

R¹² and R¹³ are a C₁₋₄ alkyl;

R is a hydrogen or a substituent selected from the group consisting ofan acyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl,methoxytrityl, tetrahydropyranyle, triphenylmethyl group, and a silylicderivative;

R¹⁶ is the HRP protein (horseradish peroxidase), the alkalinephosphatase protein, the POD protein (peroxidase) or a group of formula(III) or (IV),

The term “I¹²⁵” refers to a radio-isotope of the Iodine atom. The group(III) is bonded to the compound of formula (II) by its “amino” NHfunction. The group (IV) is bonded to the compound (II) by its “amido”C(O)NH— function. Alternatively, R¹⁶ may be ¹²⁵I-labeled histamine,¹²⁵I-labeled histidine, ¹²⁵I-labeled tyrosine, ¹²⁵I-labeled methyltyrosinate, a fluorescent group, a chemiluminescent group or a group offormula (V)

wherein Z is a linker and W is a functional group able to bind with acarbonyl group. Z may be a C₁-20 alkyl substituted or not. W may beamino, amido, hydroxyl, or hydrazine moiety.

Preferably, said tracer may be of formula (II),

in which

n is an integer between 0 and 3; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, R¹⁴, R¹⁵ are hydrogen; R¹² and R¹³ are a methyl group;

R¹⁶ is a group of formula (III).

In particular, the diagnostic device may contain a sample of a tracer offormula (II) in which n is equal to 0; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are hydrogen; R¹² and R¹³ are a methyl group; R¹⁶is a group of formula (III).

Alternatively, said means of expression of a signal is a biosensor. Thislatter expressed a signal when a monoclonal antibody binds one antigen.The term “biosensor” refers to a physicochemical device able to detect abiological sample in a physicochemical, optical, piezoelectric,electrochemical, or electromagnetic manner. The biosensor notablyincludes a related electronic element, or signal processor allowing thehandling or display of data, and a detecting element detectingphysicochemical changes in the form of signals. The monoclonal antibodymay be bonded to a support in the biosensor.

According to another aspect of the invention, the hapten of generalformula (I) as defined above, rendered immunogenic, may be used for theproduction of a hybridoma. Said production of a hybridoma may comprisethe steps of:

a) immunisation of an animal with a hapten, rendered immunogenic, offormula (I), or a salt thereof, or a derivative thereof in which thecarboxylic acid function is protected to form an ester, amide oroxazoline,

in which

n is an integer between 0 and 3;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independentlyhydrogen or a C₁₋₄ alkyl,

R¹² and R¹³ are a C₁₋₄ alkyl;

R is a hydrogen or a substituent selected from the group consisting ofacyl, benzyl, alkyl, aryl, alkyl ether, dimethoxytrityl, methoxytrityl,tetrahydropyranyle, triphenylmethyl group, and a silyl derivative; andb) sampling of B cells produced by the animal and fusion of the samewith myeloma cells to form the hybridoma. From a hybridoma obtainedaccording to this process, a process for producing a monoclonalantibody, or fragments thereof, able to recognize, or directed against,25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ may be provided.

The fact that the monoclonal antibody obtained is able to identify25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ is extremely surprisingand overcomes a technical obstacle whereby such property could not beobtained using a monoclonal antibody originating from a singlehybridoma. A significant advantage of the process of the invention isthat it allows the simultaneous recognition of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ in a sample, in a simple procedure, which is alsoeasily reproducible. In addition, this process responds to along-unfulfilled industry need, despite the fact that technology in thefield of monoclonal antibodies has been known for several decades.

DETAILED DESCRIPTION OF THE INVENTION

The present invention shall be described according to a specific form ofexecution in which the hapten, used for the immunisation of the animal,is a derivative of formula (I) in which n is an integer equal to 0; R,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are hydrogen; R¹² andR¹³ are a methyl group. This hapten shall be referred to as (H) for theremaining description. It is important to understand that the inventionis not limited to this embodiment. For example, the hapten may be aderivative of formula (I) in which n is equal to 1, 2 or 3 withsubstituents unchanged.

Procedure for the Production of a Monoclonal Antibody

The production of a monoclonal antibody may be effected following theconventional procedure such as described, for example, in Kohler andMilstein, Nature 1975 (256), 495-497 or Eur. J. Immunol. 1976 (6),511-519. According to this method, myeloid cells are combined withlymphocytic B cells of an immunised animal to obtain hybrid cells, knownas a hybridoma, which produce a monoclonal antibody. In this procedure,a hapten is bonded to a carrier protein to form an immunogen which shallbe capable of inducing immunogenicity. The carrier protein thus allowsthe hapten to gain the ability to trigger an immune reaction.

Synthesis of the Hapten and Preparation of the Immunogen

Therefore, in the present invention, the hapten (H), the synthesis ofwhich is known to those of ordinary skill in the art, is initiallycoupled with the bovine serum albumin (BSA) according to the followingprotocol. In a medium including anhydrous dimethylformamide (Fluka1386923-43408231), anhydrous dioxane (Aldrich S39136-277) anddiisopropylethylamine (Fluka 03440), an amount of hapten (H) isactivated for four hours at ambient temperature by theO—(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TSTU—Fluka 85972) dissolved in anhydrous dimethylformamide.Subsequently, 100 to 1000 hapten equivalents in solution are added to aBSA equivalent (Calbiochem 12659) diluted in carbonate buffer solution(0.1M; pH 9.4). The solution is stirred at ambient temperature for 18h-away from exposure to light. The reactional mixture is then dialysedin a saline solution NaCl (9 g/l) for 48 h at 4° C. A fresh salinesolution is re-introduced after 24 h.

The hapten (H) may also be coupled with KLH (Keyhole limpet hemocyanine,Sigma H2133). In this case, 20,000 to 500,000 equivalents of hapten insolution are added to a KLH equivalent diluted in the carbonate buffersolution (0.1M; pH 9.4). The solution is then stirred at ambienttemperature for 18 h and away from exposure to light. The reactionmixture is then dialysed in a saline solution NaCl (9 g/l) for 48 h at4° C. to obtain the desired immunogen. A fresh saline solution isre-introduced after 24 h.

Immunisation of the Animal

The female mice (6 weeks) used were provided by the CREAL company. Theimmunogen (20 μg in physiological saline solution) was injectedsubcutaneously in the mice with an additive (50% saline solution/50%additive (vol/vol)) which could be Freund's complete adjuvant (CFA,Difco Laboratories and reference: 263810) or Freund's incompleteadjuvant (IFA, Difco Laboratories and reference: 263910). The CFA is oilincluding killed mycobacteria. IFA is the same adjuvant as CFA withoutthe mycobacteria. The adjuvant allows an immune response to be triggeredin the body of the animal. After some injections of the immunogen (from3 to 10), the serum of each immunised mouse was tested to monitor theserous content in terms of specific antibodies to the injected antigen.As soon as a mouse was considered positive (namely where the percentagebinding of the radioactive-marked antigen or enzymatic marker achievedat least 10% when incubated with the mouse serum), it was selected forthe cellular fusion carried out in the month following the serum test.During the fusion, the animal was euthanized (using CO₂), its spleen wasextracted and the immunocompetent cells present in the spleen of theanimal were retrieved by rubbing the spleen with flat-tipped forcepswhile perfusing it slowly with 10 ml of medium W/O kept at 37° C.,namely a DMEM Dulbecco modified Eagle's Medium (GIBCO 21969) withoutprotein and complemented with 2%(vol/vol) of a mixture 100× ofpenicillin and streptomycin. The cells thus sampled were transferred ina Petri dish, then in a sterile conical tube of 15 ml which was thencentrifuged. The cell residue was then retrieved to be combined with themyeloid cells.

Myeloid Cells

Normally, the myeloma used for the fusion of mouse splenocytes are NSOmyeloma (Sigma ref: 85110503), SP2/0-Ag14 (ATCC ref: CRL-1581) orP3X63Ag8.653 (ATCC ref: 1580). The myeloma were cultivated in a mediumincluding a DMEM base mixture without additional protein of 2%(vol/vol)of a mixture 100× of penicillin and streptomycin, 2 to 5%(vol/vol) ofglutamine, 2%(vol/vol) of non-essential amino acids (100×), 2%(vol/vol)of sodium pyruvate (100×), 1%(vol/vol) of gentamicine. The medium alsoincluded 10%(vol/vol) of foetal calf serum. The culture was centrifugedin the sterile conical tubes of 50 ml (at 1000 rpm, 10 minutes). Thebases were gathered and centrifuged in a tube of 15 ml. Generallyspeaking, 1 ml of myeloma represents between 1 10⁶ and 2 10⁸ cells.

Cellular Fusion

The spleen cells were mixed with the myeloma cells, to form a hybridoma,with a ratio of around 5 to 10 spleen cells for 1 myeloma cell. In thepresent example: 4.7 ml of NSO myeloma at 3.4.10⁶ cells/ml were mixedwith 8.10⁷ spleen cells. The mixture of spleen cells with myeloma cellswas centrifuged and the supernatant removed. The cell residue thusobtained was slowly (1 minute) suspended in 1 ml of a polyethyleneglycol solution 50%, with the temperature maintained at 37° C. for thisoperation. The polyethylene glycol solution was obtained by dissolving 5g of polyethylene glycol (Merck, ref: 1.09727.0100) in 5 ml of phosphatebuffer 0.1 M at pH 7.4, then adding 5%(vol/vol) of dimethylsulfoxide tothe same (Sigma D2650) and sterilising by filtration (0.2 μm). The cellresidue thus re-suspended in the PEG solution was diluted at least 10times, by adding a W/O medium (DMEM without additional protein of2%(vol/vol) of a mixture 100× of penicillin and streptomycin). The tubewas centrifuged and the cell residue re-suspended in the HAT medium,including a DMEM base mixture without protein with added 2%(vol/vol) ofa mixture 100× of penicillin and streptomycin, 2 to 5%(vol/vol) ofglutamine, 2%(vol/vol) of non-essential amino acids (100×), 2%(vol/vol)of sodium pyruvate (100×), 1%(vol/vol) of gentamicine, 10%(vol/vol) offoetal calf serum, 2%(vol/vol) of hypoxanthine thymidine (50×),2%(vol/vol) of aminopterine (50×), and finally 2%(vol/vol) of NutridomaCS (Roche ref: 1363 743). The volume of HAT medium added to the cellresidue was such that the spreading over the plates of 96 vessels wasfrom 5 10⁴ to 10⁵ cells per vessel, based on 150 to 200 μl of medium pervessel. The plates were incubated for between 8 to 10 days, at 37° C.and with 5% CO₂.

Preselection of Hybridomas Each Producing a Hapten-Specific Antibody

The identification of wells from deep-well plates containing hybridomaproducing monoclonal antibody able to recognize 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ was conducted using an RIA (Radio Immuno Assay)method during the homogenous phase, followed by an immunoprecipitation.This method consists of incubating a volume (between 50 and 100 μl) ofthe culture medium originating from the deep-well plates of the fusion,with a tracer of formula (II) in which n is equal to 0; R, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are hydrogen; R¹² and R¹³ are amethyl group; R¹⁶ is a group of formula (III). After an incubationperiod of between 4 and 18 h, the reaction was stopped by the additionof SAC CEL (a cellulose suspension containing anti-mouse antibodies: IDSreference: AASAC4) to the medium. After centrifuging, the radioactivityof the bases was measured in a gamma counter. The positive wells (namelybinding more than 10% of the total added radioactivity) are those whichcontained mouse antibodies specifically for the hapten (H), hence thosecontaining specific hybridoma clones.

Selection of Hybridoma Clones Producing a Monoclonal AntibodyRecognizing Both 25-Hydroxyvitamin D₂ and 25-Hydroxyvitamin D₃

Among the positive hybridomas (namely those producing monoclonalantibodies which identified the tracer) obtained, those identifying both25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were selected by aninhibition test of the binding of said tracer. This test consisted ofincubating the culture's supernatant (for example 100 μl) of thehybridoma (in a Petri dish) with the tracer (marked with iodine 125) andin the presence of 25-hydroxyvitamin D₂ or 25-hydroxyvitamin D₃. Themixture was incubated at ambient temperature for 18 h. The25-hydroxyvitamin D₂ used was provided by Sigma under reference H17937,while the 25-hydroxyvitamin D₃ was provided by Sigma under referenceH4014. The recognition tests were carried out with solutions of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ at concentrations of 1000ng/ml. The tracer was in solution in a mixture volume/volume: 25%water/25% ethanol/50% phosphate buffer 0.1M pH 7.4 CP (casein peptone:organotechnical reference: 1 9516) 10 g/l. The reaction was stopped byimmunoprecipitation by adding SAC CEL (cellulose suspension containinganti-mouse antibodies: IDS reference: AASAC4) to the incubation medium.

Results

Table 1 represents the recognition results of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ for selected hybridoma clones followingpreselection tests of hybridomas producing hapten-specific monoclonalantibodies.

TABLE 1 Without Vit. 25-OH-Vitamin D₂ 25-OH-Vitamin D₃ D₂ in Vit. D₃ 1μg/ml 1 μg/ml Col 1 Col 2 Col 3 Col 4 Col 5 Col 6 Clone No. Fixation (%)Fixation (%) Inhibition (%) Fixation (%) Inhibition (%) LMBP 7011CB 63.247.2 25.3 9.4 85.2 LMBP 7012CB 60.0 10.0 83.3 0.9 98.5 LMBP 7013CB 48.630.6 36.9 18.3 62.3 LMBP7204CB 57.1 23.6 58.6 28.5 50.1 LMBP7205CB 68.742.0 38.0 51.8 24

The first column re presents the numbers of the tested clones, whichcorrespond to the hybridoma deposited as described above. The secondcolumn corresponds to the percentage of binding of said tracer to themonoclonal antibody produced by the corresponding clone, in the absenceof 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. The third columncorresponds to the percentage of binding of said tracer to themonoclonal antibody produced by the corresponding clone in the presenceof 25-hydroxyvitamin D₂ at a concentration in solution of 1 μg/ml. Thefourth column corresponds to the inhibition percentage of correspondingmonoclonal antibody in the presence of 25-hydroxyvitamin D₂ at aconcentration of 1 μg/ml. This percentage is calculated by deductingfrom 100% the ratio, multiplied by 100, between the percentage ofbinding in the presence of 25-hydroxyvitamin D₂ at 1000 ng/ml and thepercentage of binding without 25-hydroxyvitamin D₂. The fifth columncorresponds to the percentage of binding of said tracer to themonoclonal antibody produced by the corresponding clone in the presenceof 25-hydroxyvitamin D₃ at a concentration in solution of 1 μg/ml. Thesixth column corresponds to the inhibition percentage of thecorresponding monoclonal antibody in the presence of 25-hydroxyvitaminD₃ at a concentration of 1 μg/ml. This percentage is calculated bydeducting from 100% the ratio, multiplied by 100, between the percentageof binding in the presence of 25-hydroxyvitamin D₃ at 1 μg/ml and thepercentage of binding without 25-hydroxyvitamin D₃.

When the concentrations of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃are around 1 μg/ml, the inhibition percentage of the monoclonal antibodyproduced by the clone LMBP7012CB may exceed 80% vis-à-vis25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃.

Example of the Simultaneous Recognition of 25-Hydroxyvitamin D₂ and25-Hydroxyvitamin D₃

A test of the simultaneous recognition of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ was conducted on coated tubes. The monoclonalantibody was anchored in dry tubes (direct coating) at a concentrationof 0.5 μg/ml. The antibody used was produced by the LMBP hybridoma7013CB. Subsequently, 300 μl of incubation buffer (phosphate 50 mM pH7.4casein peptone 2 g/l of sodium azide 0.5 g/l) and 100 μl of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were added to the tubes.The solutions were analysed after two hours at ambient temperature.

TABLE 2 Total detected Percentage of D₃ (ng/mL) D₂ (ng/mL) quantity(ng/mL) recognition (%) 1.5 1.5 3.3 110 5 5 8.1 80 15 15 25.1 84 50 5099.7 100 50 5 60 110 5 50 42 75The columns titled “D₃” and “D₂” in Table 2, respectively correspond tothe concentrations, expressed in ng/mL, of 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ used for the recognition test. For example, whenthe concentrations of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ wererespectively 50 ng/ml, more than 99 ng/ml of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ was detected, which represents a particularlynoteworthy result.

Identical tests were carried out with the monoclonal antibody producedby the LBMP hybridoma 7012CB. Therefore, when the concentrations in25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were each 1.5 ng/mL, therecognition percentage w as 86%. Similarly, when the concentrations of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were respectively 5 ng/mL,the recognition percentage was 77%. Excellent results were also obtainedfor other concentrations. Tests were also carried on with a monoclonalantibody produced from hybridomas LMBP 7204CB or LMBP 7205CB. When theconcentrations in 25-hydroxyvitamin D₂ et 25-hydroxyvitamine D₃ wereeach 1,5 ng/mL, the recognition percentage was 104% with a monoclonalantibody from the hybridoma LMBP 7204CB. When the concentration in25-hydroxyvitamin D₂ was 5 ng/mL and the concentration in25-hydroxyvitamine D₃ was 50 ng/mL, recognition percentage was 100% witha monoclonal antibody from the hybridoma LMBP 7205CB. These two clonesshow an excellent recognition of 25-hydroxyvitamin D₂. The tests carriedout with the monoclonal antibody produced by the hybridoma LMBP 7011CBalso revealed good results.

Testing was also performed in human serum from patient. After apre-treatment allowing the releasing of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃, the simultaneous recognition of 25-hydroxyvitaminD₂ and 25-hydroxyvitamin D₃ was conducted on coated tubes where themonoclonal antibody was anchored in dry tubes (indirect coating) at aconcentration of 25 ng/ml. In table 3, the monoclonal antibody used wasproduced by the LMBP hybridoma 7013CB. In table 4, the monoclonalantibody used was produced by the LMBP hybridoma 7012CB. A referencemethod, LC-MS (Liquid Chromatography-Mass Spectrometry) has been used tovalidate the concentrations found in the human serum.

TABLE 3 [25OH Vit. D₂ + [25OH Vit. D₂ + 25OHVit. D₃] [25OH Vit. D₂][25OH Vit. D₃] 25OH Vit. D₃] Human ng/ml with Mab ng/ml ng/ml ng/mlRecognition serum from 7013CB with LC-MS with LC-MS with LC-MSpercentage 1 50.3 10.7 48.0 58.7 86% 2 91.4 124.2 0 124.2 74% 3 52.314.3 47.8 62 84% 4 120 28.0 95.6 123.6 97%

TABLE 4 [25OH Vit D₂ + [25OH Vit D₂ + 25OH Vit D₃] [25OH Vit D₂] [25OHVit D₃] 25OH Vit D₃] Human ng/ml with Mab ng/ml ng/ml ng/ml Recognitionserum from 7012CB with LC-MS with LC-MS with LC-MS percentage 5 42.8 4.138.4 42.5 101% 6 20.9 6.8 13.6 20.4 102% 7 43.1 10.5 36.8 47.3  91%Rat serum and mouse serum have been tested using the same protocol.Similar results were obtained.

Obtaining of Monoclonal Antibody

Following the selection of the clones of interest, the cells were frozenin liquid nitrogen for long-term preservation. The production of amonoclonal antibody is performed by a system of in vitro culture ofhybridoma cells for example: in a spinner flask (Wheaton Magna-Flex®Microcarrier Spinner Flasks), or in CELLINE (Integra Bioscience) or anyother method of in vitro culture suitable for hybridoma cells.Alternatively, the monoclonal antibody can be produced by an in vivomethod, such as that featuring ascites, where permitted by law.

Purification of the Monoclonal Antibody

The culture's supernatant originating from the in vitro culture systemof hybridoma cells is purified by affinity chromatography on aconventional column of protein A and/or protein G (GE Healthcare), on aSTREAMLINE Protein A support (GE Healthcare).

Diagnostic Devices

The diagnostic devices according to the invention, also known asimmunoassay devices can include “enzyme immunoassays” devices (EIA) or“enzyme-linked immunosorbent assays” devices (ELISA), an“immunofluorescence” device (IFA), a radiometric device or“radioimmunoassays” (RIA), a “magnetic separation assays” device (MSA),a “lateral flow assays” device, a “diffusion immunoassays” device, animmunoprecipitation device, an “immunosorbent” or “antigen-down assays”device, an immuno-agglutination device, a “chemilunescence immuno assay(CLIA)” device or also a device using a biosensor.

Various types of supports can be used: tubes, microtitration plates orblocks. The monoclonal antibody or the fragments thereof can bebiotinylated to increase the binding or sensitivity to the differentsupports.

The monoclonal antibody, or fragments related to the same may be bounddirectly onto the support. It is also possible to bind an anti-mouseantibody to the support, followed by monoclonal antibody or fragmentsthereof according to the invention on this initial antibody. Themonoclonal antibody or fragments thereof should preferably bebiotinylated.

Alternatively, the hapten may be bound to the support. Subsequently, amonoclonal antibody according to the invention is added, followed by ananti-HRP secondary antibody. If required, the monoclonal antibody orfragments thereof can be used by direct binding to the HRP (horse radishperoxidase) in order to detect the immunogenic bound beforehand onto asupport. Alternatively, the monoclonal antibody, or the fragmentsrelated to the same, can be biotinylated. Subsequently, the SAv-HRP(streptavidin-horse radish peroxidase) is added.

The tracer may be bound to a tyramine marked with iodine 125 for the RIAtests. It may also be used after biotinylation or binding to the HRPwith a view to conducting ELISA or CLIA tests. The luminol ortetramethylbenzidine can subsequently be bonded to the HRP.

Finally, the tests can be performed in open or sealed automatic units.

In the competitive RIA tests, a fixed quantity of the tracer of formula(II) in which n is equal to 0; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, R¹⁴, R¹⁶ are hydrogen; R¹² and R¹³ are a methyl group; R¹⁶ is agroup of formula (III), starts competing with the 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ present in the samples, controls or calibratorsextracted for a fixed quantity of specific antibody, immobilised on thesurface of a polystyrene support. After an incubation of 2 to 24 h atambient temperature or at 37° C., an aspiration phase ends thecompetitive reaction. The tubes are then washed and the radioactivity ismeasured in a gamma counter.

Thanks to the present invention, the recognition of 25-hydroxyvitamin D₃and 25-hydroxyvitamin D₂ is simultaneous when using diagnostic devicesaccording to the Invention on samples containing 25-hydroxyvitamin D₃and 25-hydroxyvitamin D₂.

The terms and descriptions used here are solely intended to be forreference and are not intended to be exhaustive. It is generallyrecognished that numerous variations are possible in the vein andbreadth of the invention such as described in the claims which followand their equivalents; in which all the terms must be understand as partof wider acceptance unless otherwise specified.

1. A process for the production of a hybridoma, and of a monoclonalantibody or fragments thereof, able to recognize 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ comprising the following steps: a) immunisationof an animal with a hapten, rendered immunogenic, of formula (1), or asalt thereof, or a derivative thereof in which the carboxylic acidfunction is protected to form an ester, amide or oxazoline,

in which n is an integer between 0 and 3; R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independently hydrogen or a C₁₋₄ alkyl, R¹²and R¹³ are a C₁₋₄ alkyl; R is a hydrogen or a substituent selected fromthe group consisting of acyl, benzyl, alkyl, aryl, alkyl ether,dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethylgroup, and a silyl derivative; b) sampling of B cells produced by theanimal and fusion of the same with myeloma cells to form the hybridoma;c) production, from at least part of the obtained hybridoma, of amonoclonal antibody, or fragments thereof, able to recognize25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃.
 2. The process accordingto claim 1, wherein the recognition, by said monoclonal antibody, of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ is simultaneous when bothantigens are in the same sample.
 3. The process according to claim 1,wherein said monoclonal antibody has a recognition percentage of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ ranging from 70 and 110%.4. The process according to claim 1, wherein the hapten is thederivative of formula (1) in which n is equal to 0; R, R², R³, R⁴, R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are respectively hydrogen; R¹² andR¹³ are respectively methyl group.
 5. The process according to claim 1,wherein the hybridoma is selected from the group consisting of thehybridomas deposited in the BGGM/LMBP under deposit numbers LMBP 7011GB,LMBP 7012GB and LMBP 7013GB, LMBP 7204GB and LMBP 7205GB.
 6. The processaccording to claim 1, wherein the hapten is rendered immunogenic by thecovalent coupling between the hapten and a carrier protein, said bindingenabled via the carboxylic acid function of said hapten.
 7. A hybridomasuitable for the production of a monoclonal antibody or fragmentsthereof able to recognize 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃.8. The hybridoma according to claim 7, wherein it is selected from thegroup consisting of the hybridomas deposited in the BGGM/LMBP underdeposit numbers LMBP 7011GB, LMBP 7012GB, LMBP 7013GB, LMBP 7204GB andLMBP 7205GB.
 9. A monoclonal antibody or fragments thereof able torecognize 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in a sample. 10.The monoclonal antibody according to claim 9, wherein one or more of thefollowing conditions are met: a) the recognition of 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ is simultaneous when both antigens are in thesame sample; and b) the recognition percentage of 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃ is between 70 and 110%.
 11. (canceled)
 12. Themonoclonal antibody according to claim 9, wherein it is produced from ahybridoma selected from the group consisting of the hybridomas depositedin the BGGM/LMBP under deposit numbers LMBP 7011GB, LMBP 7012GB, LMBP7013GB, LMBP 7204GB and LMBP 7205GB.
 13. A method of recognition ofand/or quantitation of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ ina sample comprising the steps of: providing a biological sample to betested; testing the sample on a diagnostic device, wherein thediagnostic device uses a hapten consisting of a compound of formula (1),or a salt thereof, or a derivative thereof in which the carboxylic acidfunction is protected to form an ester, amide or oxazoline,

in which n is an integer between 0 and 3; R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independently hydrogen or a C₁₋₄ alkyl; R¹²and R¹³ are a C₁₋₄ alkyl; R is a hydrogen or a substituent selected fromthe group consisting of acyl, benzyl, alkyl, aryl, alkyl ether,dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethylgroup, and a silyl derivative; said hapten rendered immunogenic; for theproduction of a hybridoma, and of a monoclonal antibody, or fragments orrecombinant fragments thereof, able to recognize 25-hydroxyvitamin D₂and 25-hydroxyvitamin D₃.
 14. The method according to claim 13, whereinsaid hapten is rendered immunogenic by the binding between the haptenand a carrier protein, said binding enabled via the carboxylic acidfunction of said hapten.
 15. The method according to claim 13, whereinsaid hapten of formula (1) is the compound of formula (1) in which n isequal to 0; R, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ arerespectively hydrogen; R¹² and R¹³ are respectively methyl group. 16.The method according to claim 13, wherein said monoclonal antibody, orthe fragments thereof, produced is a monoclonal antibody capable of thesimultaneous recognition of 25-hydroxyvitamin D₂ and 25-hydroxyvitaminD₃ when both antigens are in the same sample.
 17. A method ofrecognition of and/or quantitation of 25-hydroxyvitamin D₂ and25-hydroxyvitamin D₃ in a sample comprising the steps of: providing abiological sample to be tested; testing the sample on a diagnosticdevice, wherein the diagnostic device uses a hapten consisting of acompound of formula (1), or a salt thereof, or a derivative thereof inwhich the carboxylic acid function is protected to form an ester, amideor oxazoline,

in which n is an integer between 0 and 3; R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independently hydrogen or a C₁₋₄ alkyl, R¹²and R¹³ are a C₁₋₄ alkyl; R is a hydrogen or substituent selected fromthe group consisting of acyl, benzyl, alkyl, aryl, alkyl ether,dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethylgroup, and a silyl derivative; said hapten rendered immunogenic.
 18. Adiagnostic device able to allow the recognition and/or quantification of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in a sample to be testedcomprising the monoclonal antibody able to recognize 25-hydroxyvitaminD₂ and 25-hydroxyvitamin D₃ of claim
 9. 19. A diagnostic deviceaccording to claim 18, wherein it further comprises a sample of thetracer of formula (II),

in which n is an integer between 0 and 3; R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independently hydrogen or a C₁₋₄ alkyl, R¹²and R¹³ are a C₁₋₄ alkyl; R is a hydrogen or a substituent selected fromthe group consisting of acyl, benzyl, alkyl, aryl, alkyl ether,dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethylgroup, and a silyl derivative; R¹⁶ is the HRP protein (horseradishperoxidase), the alkaline phosphatase protein, the POD protein(peroxidase), a group of formula (III) or (IV), ¹²⁵I-labeled histamine,¹²⁵I-labeled histidine, ¹²⁵I-labeled tyrosine, ¹²⁵I-labeled methyltyrosinate, a fluorescent group, a chemiluminescent group or a group offormula (V) wherein Z is a linker and W is a functional group able tobind with a carbonyl group.


20. A diagnostic device according to claim 18, wherein it is selectedfrom the group consisting of enzyme immunoassays (EIA) device,enzyme-linked immunosorbent assays (ELISA) device, an immunofluorescenceassay (IFA) device, a radioimmunoassay device (RIA), a “magneticseparation assays” device (MSA), a “lateral flow assays” device, a“diffusion immunoassays” device, an immune-precipitation device, an“immunosorbent” or “antigen-down assays” device an immune-agglutinationdevice, a “chemilunescence immune assay (CLIA) device, or a device usinga biosensor.
 21. A method for the production of a hybridoma comprisingthe steps of: immunisation of an animal or human with a hapten, renderedimmunogenic, of general formula (1), or a salt thereof, or a derivativethereof in which the carboxylic acid function is protected to form anester, amide or oxazoline,

in which n is an integer between 0 and 3; R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵ are independently hydrogen or a C₁₋₄ alkyl, R¹²and R¹³ are a C₁₋₄ alkyl; R is a hydrogen or a substituent selected fromthe group consisting of acyl, benzyl, alkyl, aryl, alkyl ether,dimethoxytrityl, methoxytrityl, tetrahydropyranyle, triphenylmethylgroup, and a silyl derivative; and sampling of B cells produced by theanimal or human and fusion of the same with myeloma cells to form thehybridoma.